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Viral paratransgenesis in the malaria vector Anopheles gambiae.

Ren X, Hoiczyk E, Rasgon JL - PLoS Pathog. (2008)

Bottom Line: To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes.Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations.AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae.

View Article: PubMed Central - PubMed

Affiliation: The Johns Hopkins Malaria Research Institute, Baltimore, Maryland, United States of America.

ABSTRACT
Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms, is being considered as a potential method to control vector-borne diseases such as malaria. The feasibility of paratransgenic malaria control has been hampered by the lack of candidate symbiotic microorganisms for the major vector Anopheles gambiae. In other systems, densonucleosis viruses (DNVs) are attractive agents for viral paratransgenesis because they infect important vector insects, can be genetically manipulated and are transmitted to subsequent generations. However, An. gambiae has been shown to be refractory to DNV dissemination. We discovered, cloned and characterized the first known DNV (AgDNV) capable of infection and dissemination in An. gambiae. We developed a flexible AgDNV-based expression vector to express any gene of interest in An. gambiae using a two-plasmid helper-transducer system. To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes. Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations. These proof-of-principle data suggest that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-Plasmodium peptides or insect-specific toxins in Anopheles mosquitoes. AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae.

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Related in: MedlinePlus

EGFP expression in An. gambiae F1 adults infected with wild-type AgDNV (from pBAgα) and EGFP-transducing virions (from pAgActinGFP), which demonstrates transmission of transducing virions between mosquito generations.A–C: EGFP expression in abdomen, D: EGFP expression in head, E: EGFP expression in maxillary palp.
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ppat-1000135-g008: EGFP expression in An. gambiae F1 adults infected with wild-type AgDNV (from pBAgα) and EGFP-transducing virions (from pAgActinGFP), which demonstrates transmission of transducing virions between mosquito generations.A–C: EGFP expression in abdomen, D: EGFP expression in head, E: EGFP expression in maxillary palp.

Mentions: EGFP-positive mosquitoes were allowed to reproduce. We observed EGFP expression in approximately 20% of F1 offspring (N>50, Figure 8) and detected EGFP DNA by PCR and sequencing from EGFP-expressing F1 mosquitoes (N = 8). We continued to breed the offspring and again assessed EGFP expression in the F3 generation, where 20% of the mosquitoes had observable EGFP fluorescence (N = 20). These data indicate that AgDNV can be used to drive expression of exogenous transgenes in An. gambiae and that transducing virions are transmitted to subsequent generations, similar to wild-type virus. While it is not clear at this point whether offspring are infected by transovarial transmission or horizontal transmission from adults to larvae, we detected EGFP in both developing ovarioles and in mature oocytes (Figure 7) suggesting that transovarial transmission may be involved.


Viral paratransgenesis in the malaria vector Anopheles gambiae.

Ren X, Hoiczyk E, Rasgon JL - PLoS Pathog. (2008)

EGFP expression in An. gambiae F1 adults infected with wild-type AgDNV (from pBAgα) and EGFP-transducing virions (from pAgActinGFP), which demonstrates transmission of transducing virions between mosquito generations.A–C: EGFP expression in abdomen, D: EGFP expression in head, E: EGFP expression in maxillary palp.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2500179&req=5

ppat-1000135-g008: EGFP expression in An. gambiae F1 adults infected with wild-type AgDNV (from pBAgα) and EGFP-transducing virions (from pAgActinGFP), which demonstrates transmission of transducing virions between mosquito generations.A–C: EGFP expression in abdomen, D: EGFP expression in head, E: EGFP expression in maxillary palp.
Mentions: EGFP-positive mosquitoes were allowed to reproduce. We observed EGFP expression in approximately 20% of F1 offspring (N>50, Figure 8) and detected EGFP DNA by PCR and sequencing from EGFP-expressing F1 mosquitoes (N = 8). We continued to breed the offspring and again assessed EGFP expression in the F3 generation, where 20% of the mosquitoes had observable EGFP fluorescence (N = 20). These data indicate that AgDNV can be used to drive expression of exogenous transgenes in An. gambiae and that transducing virions are transmitted to subsequent generations, similar to wild-type virus. While it is not clear at this point whether offspring are infected by transovarial transmission or horizontal transmission from adults to larvae, we detected EGFP in both developing ovarioles and in mature oocytes (Figure 7) suggesting that transovarial transmission may be involved.

Bottom Line: To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes.Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations.AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae.

View Article: PubMed Central - PubMed

Affiliation: The Johns Hopkins Malaria Research Institute, Baltimore, Maryland, United States of America.

ABSTRACT
Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms, is being considered as a potential method to control vector-borne diseases such as malaria. The feasibility of paratransgenic malaria control has been hampered by the lack of candidate symbiotic microorganisms for the major vector Anopheles gambiae. In other systems, densonucleosis viruses (DNVs) are attractive agents for viral paratransgenesis because they infect important vector insects, can be genetically manipulated and are transmitted to subsequent generations. However, An. gambiae has been shown to be refractory to DNV dissemination. We discovered, cloned and characterized the first known DNV (AgDNV) capable of infection and dissemination in An. gambiae. We developed a flexible AgDNV-based expression vector to express any gene of interest in An. gambiae using a two-plasmid helper-transducer system. To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes. Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations. These proof-of-principle data suggest that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-Plasmodium peptides or insect-specific toxins in Anopheles mosquitoes. AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae.

Show MeSH
Related in: MedlinePlus